Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media. Adjust volumes accordingly.
To 800ml ddH2O add:
- 8 g NaCl
- 0.2 g KCl
- 1.44 g Na2HPO4 (sodium phosphate dibasic)
- 0.24 g KH2PO4 (monopotassium phosphate)
pH to 7.4, and bring up to 1L with ddH2O. Autoclave to sterilize.
Saturated Ammonium Sulphate (SAS):
To 1000 ml of double-distilled water add:
- 766.8 g g Ammonium sulphate<ref>http://cshprotocols.cshlp.org/content/2006/1/pdb.tab1</ref><ref>http://research.biology.arizona.edu/mosquito/willott/proj/LabPro/Prot/Proppt/Ammon.html</ref><ref>http://www.science.smith.edu/departments/Biochem/Biochem_353/Amsulfate.htm</ref>
Dissolve at 50C to 60C. Stand overnight at room temperature, then adjust pH to 6.8-7.0 with 5 or 10 M NaOH. Measure the pH using small aliquots.
Per 100 ml of water add:
- 355 mg (20mM) sodium phosphate dibasic
- 292 mg (10mM) EDTA
pH to 7.0. Immediately before use add 20mM cysteine-HCl
To 100ml of water add
- 158 mg Tris-HCl
pH to 7.5.
- Dilute antibody 1:2 with PBS on ice. E.G. 10ml of hybridoma media would be diluted with 20ml of PBS.
- With gentle stirring, on-ice, add SAS drop-wise to reach a final concentration of 45% ammonium hydroxide (see calculator below). Warning: stir gently. Avoid foaming, as this indicates protein denaturation.
- Once SAS addition is complete stir for 30min on-ice, then centrifuge at 1000g for 15 min at 4°C.
- Wash the precipitate with 45% SAS, then re-pellet at 1000g for 15 min at 4°C.
- Redissolve the precipitate in the same volume of PBS as the original antibody solution.
- Centrifuge at 5000g for 15 min at 4°C to remove any particulates.
- Transfer the supernatant to a clean tube and reprecipitate immunoglobulin using 40% SAS
- Centrifuge at 1000g for 15 min at 4°C.
- Redissolve the precipitate in a minimum volume of digestion buffer without cysteine (0.5 mL) and dialyse against 5L of digestion buffer without cysteine at 4°C overnight.
- Centrifuge at 5000g for 15 min at 4°C to remove more insoluble material.
- Spec the protein to determine the concentration.
SAS Volume Calculator:
For 1ml of antibody solution add:
File:SAS Calc.gif, where
SF = final saturation, Si = initial saturation. Example, for 45% of a 1ml starting volume with no prior SAS addition:
Volume (ml) = 1(0.45-0) / (1-0.45) = 0.82ml
- Add 20mM cystein-HCl to the fractionated IgG & digest buffer.
- Dilute fractionated IgG to a concentration of 10mg/ml in digest buffer.
- Mix immobilized papain beads (Thermo #20341) to obtain an even suspension. Take 500ul of slurry* for every 1ml of diluted IgG to be processed. Transfer slurry to a clean tube large enough to perform the digest in.
- Wash gel 2X in 4ml/500ul of slurry with digest buffer. Centrifuge briefly (1-2 min at 250 x g) to pellet papain.
- Pellet washed papain, and resuspend in 0.5ml of digest buffer per 500ul of starting slurry.
- Add antibody solution to papain slurry.
- For non-human IgG, incubate 5 hours to overnight at 37oC with mixing. Four hours is sufficient for human IgG.
- For every 1ml of diluted antibody add 1.5ml 10mM tris-HCl solution, then centrifuge 1000g for 15 min at 4°C.
- Carefully draw supernatant off of papain pellet.
- Smaller amounts of papain may be used, but longer digests are required and incomplete digestion may occur as a result. For antibody quantities less than 10mg, use 500ul of slurry.
Fab purification should be performed as per the instructions found in Fab purification using FPLC.